3'-processing and strand transfer catalysed by retroviral integrase in crystallo

Retroviral integrase (IN) is responsible for two consecutive reactions, which lead to insertion of a viral DNA copy into a host cell chromosome. Initially, the enzyme removes di- or trinucleotides from viral DNA ends to expose 3'-hydroxyls attached to the invariant CA dinucleotides (3'-processing reaction). Second, it inserts the processed 3'-viral DNA ends into host chromosomal DNA (strand transfer). Herein, we report a crystal structure of prototype foamy virus IN bound to viral DNA prior to 3'-processing. Furthermore, taking advantage of its dependence on divalent metal ion cofactors, we were able to freeze trap the viral enzyme in its ground states containing all the components necessary for 3'-processing or strand transfer. Our results shed light on the mechanics of retroviral DNA integration and explain why HIV IN strand transfer inhibitors are ineffective against the 3'-processing step of integration. The ground state structures moreover highlight a striking substrate mimicry utilized by the inhibitors in their binding to the IN active site and suggest ways to improve upon this clinically relevant class of small molecules.     

Dissimilarity-based algorithms for selecting structurally diverse sets of compounds.

This paper commences with a brief introduction to modern techniques for the computational analysis of molecular diversity and the design of combinatorial libraries. It then reviews dissimilarity-based algorithms for the selection of structurally diverse sets of compounds in chemical databases. Procedures are described for selecting a diverse subset of an entire database, and for selecting diverse combinatorial libraries using both reagent-based and product-based selection.     

DNA strand transfer reactions catalyzed by vaccinia topoisomerase I.

Vaccinia virus DNA topoisomerase I forms a 3'-phosphoryl intermediate with duplex DNAs containing the conserved binding/cleavage motif 5'CCCTT decreases. Covalently bound enzyme is capable of transferring the incised DNA strand to a heterologous DNA acceptor containing a 5'OH terminus. Both intramolecular and intermolecular religation reactions are catalyzed. Intramolecular strand transfer occurs to the noncleaved strand of the DNA duplex and results in formation of a hairpin loop. Intermolecular religation to an exogenous DNA strand is favored over hairpin formation and requires the potential for base pairing between the acceptor and the noncleaved strand of the donor complex. As few as 4 potential base pairs are sufficient to support intermolecular transfer. These results in vitro are consistent with the proposal that vaccinia topoisomerase can catalyze sequence-specific strand transfer during genetic recombination in vivo (Shuman, S. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 10104-10108.).     

Suppression of tropomyosin synthesis, a common biochemical feature of oncogenesis by structurally diverse retroviral oncogenes.

To identify proteins whose production may be altered as a common event in the expression of structurally diverse oncogenes, we compared two-dimensional electropherograms of newly synthesized proteins from NIH/3T3 cell lines transformed by a variety of retroviral oncogenes, from cellular revertant lines, and from a line (433.3) which expresses the v-ras oncogene in response to corticosteroids. Most alterations in the synthesis of specific proteins detected by this approach appeared to be the result of selection during prolonged cultivation and were probably unrelated to the transformation process. However, we detected seven proteins whose synthesis was strongly suppressed in cell lines transformed by each of the six retroviral oncogenes we studied and whose production was fully or partially restored in two cellular revertant lines. Suppression of two of these proteins was also correlated with the initial appearance of morphological alteration during corticosteroid-induced oncogene expression in 433.3 cells. These proteins (p37/4.78 and p41/4.75) were identified as tropomyosins, a group of at least five cytoskeletal proteins. Transformation by the papovaviruses simian virus 40 and polyomavirus caused no suppression of synthesis of these tropomyosins. This indicates that suppression of tropomyosin synthesis is not a nonspecific response by cells to being forced to grow with the transformed phenotype but is specifically associated with oncogenesis by diverse retroviral oncogenes. The results are consistent with the hypothesis that the different biochemical processes initiated by expression of structurally diverse retroviral oncogenes may converge on a limited number of common targets, one of which is the mechanism which regulates the synthesis of tropomyosins.     

Tyr60 variants of Flp recombinase generate conformationally altered protein-DNA complexes. Differential activity in full-site and half-site recombinations

The tyrosine at position 60 of the Flp recombinase of the Saccharomyces cerevisiae plasmid, 2 mu circle, is invariant among site-specific recombinases of the "yeast plasmid family". Alterations of this residue give rise to Flp variants that show no recombination activity when assayed in vivo in Escherichia coli. Upon purification, they bind substrate, execute DNA cleavage and catalyze recombination. The efficiency of strand cleavage follows the order: Flp(Y60F) greater than Flp greater than Flp(Y60S) greater than Flp(Y60D); efficiency of recombination between Flp sites on a linear substrate and a circular one follows the order: Flp greater than Flp(Y60F) greater than Flp(Y60S) greater than Flp(Y60D). Methylation footprints of the DNA-protein complexes formed by two of the Flp variants, Flp(Y60S) and Flp(Y60D), do not show hypermethylation of the G residues within the substrate core that is characteristic of complexes formed by wild-type Flp. The third variant, Flp(Y60F), causes significant distortion (although less than wild-type Flp) of the substrate core, as indicated by enhanced G-methylation. Binding profiles with circularly permuted substrates indicate that Flp(Y60S) and Flp(Y60D), but not Flp(Y60F), are defective in bending substrate DNA. In recombination between two Flp half-sites, the variant proteins are significantly more active than in normal full-site recombination.     

Bending-incompetent variants of Flp recombinase mediate strand transfer in half-site recombinations: role of DNA bending in recombination.

One key feature of the interaction of Flp recombinase with its target site (FRT) is the large bend introduced in the substrate as a result of protein binding. The extent of bending was found to depend on the phasing and spacing of the Flp monomers occupying the two Flp-binding elements (FBE) bordering the strand-exchange region (spacer) of the substrate. The relative mobilities of the Flp complexes formed by the two permuted substrate fragments, containing the FRT site near the end or in the middle, corresponded to a DNA bend of approx. 140 degrees when each of the two FBEs flanking the spacer was occupied by a protein monomer. The estimated bend angle was the same when the reference DNA fragment with the FRT site at the end was substituted by one with the site in the middle, but containing a 4-bp insertion within the spacer. We used a combination of wild-type Flp and Flp variants that were competent or incompetent in DNA bending, together with full, or half FRT sites, to ask whether bending is a conformational requirement for catalysis, namely cleavage and exchange of strands. We obtained the following results: in full-site (FRT) vs. full-site recombinations or in full-site vs. half-site (half FRT) recombinations, there was a large difference in the reactivity between Flp and a bending-incompetent Flp variant. This difference virtually disappeared when reactions were done with half-FRT sites. We conclude that bending is not a prerequisite for catalysis, but represents the manner in which the substrate accommodates the Flp protomer-protomer interactions that are pertinent to catalysis.     

Utilisation of structurally diverse organophosphonates by Streptomycetes

A group of streptomycete strains was found able to utilise a wide range of structurally diverse phosphonates as a sole phosphorus source. No relation could be observed between ability to synthesise compounds containing a direct carbon-to-phosphorus (C-P) bond and biodegradative potential towards phosphonates in the strains studied. Streptomyces morookaensis DSM 40565 could degrade 2-amino-4-phosphonobutyrate as a sole nitrogen and phosphorus source in a stereoselective-like manner. This result suggests the existence of a new metabolic pathway for C-P bond breakage.     

Syntheses of novel antimycobacterial combinatorial libraries of structurally diverse substituted pyrimidines by three-component solid-phase reactions

A new pyrimidine based scaffold has been developed by three-component solid-phase syntheses. The utility of scaffolds was demonstrated by synthesizing libraries of 80 substituted pyrimidines (a-p), (a-p), 14(a-p), 15(a-p), 16(a-p). Among 80 compounds screened, six compounds, 12i, 13c, 14d, 14e, 14o, and 15d showed in vitro activity against Mycobacterium tuberculosis (MABA) at a concentration of 50 and 25 microg/mL     

Activation of kinase pathways in MCF-7 cells by 17beta-estradiol and structurally diverse estrogenic compounds

17beta-Estradiol (E2) activates non-genomic pathways in MCF-7 cells, and this study investigates the effects of structurally-diverse estrogenic compounds on activation of mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase (PI3-K), protein kinase C (PKC), PKA, and calcium calmodulin-dependent kinase IV (CaMKIV). Activation of kinases was determined by specific substrate phosphorylation and transactivation assays that were diagnostic for individual kinases. The compounds investigated in this study include E2, diethylstilbestrol (DES), the phytoestrogen resveratrol, and the following synthetic xenoestrogens, bisphenol-A (BPA), nonylphenol, octylphenol, endosulfan, kepone, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), and 2',3',4',5'-tetrachloro-4-biphenylol (HO-PCB-Cl(4)). With the exception of resveratrol, all the compounds activated PI3-K and MAPK. Activation of PKC by the xenoestrogens was structure-dependent since resveratrol, kepone and HO-PCB-Cl(4) were inactive and only minimal activation of PKA was observed. CaMKIV was activated only by E2 and DES, and HO-PCB-Cl(4) was a potent inhibitor of CaMKIV-dependent activity. These results demonstrate that activation of estrogen receptor-alpha-mediated non-genomic pathways by estrogenic compounds in MCF-7 cells is structure-dependent and can result in activation or inhibition of kinase activities.     

DNA strand transfer reactions catalyzed by vaccinia topoisomerase: hydrolysis and glycerololysis of the covalent protein-DNA intermediate.

Vaccinia topoisomerase forms a covalent protein-DNA intermediate at sites containing the sequence 5'-CCCTT. The T nucleotide is linked via a 3'-phosphodiester bond to Tyr-274 of the enzyme. Here, we report that the enzyme catalyzes hydrolysis of the covalent intermediate, resulting in formation of a 3'-phosphate-terminated DNA cleavage product. The hydrolysis reaction is pH-dependent (optimum pH = 9.5) and is slower, by a factor of 10(-5), than the rate of topoisomerase-catalyzed strand transfer to a 5'-OH terminated DNA acceptor strand. Mutants of vaccinia topoisomerase containing serine or threonine in lieu of the active site Tyr-274 form no detectable covalent intermediate and catalyze no detectable DNA hydrolysis. This suggests that hydrolysis occurs subsequent to formation of the covalent protein-DNA adduct and not via direct attack by water on DNA. Vaccinia topoisomerase also catalyzes glycerololysis of the covalent intermediate. The rate of glycerololysis is proportional to glycerol concentration and is optimal at pH 9.5.     

Inhibition profiling of human carbonic anhydrase II by high-throughput screening of structurally diverse, biologically active compounds

Human carbonic anhydrase II (CA II), a zinc metalloenzyme, was screened against 960 structurally diverse, biologically active small molecules. The assay monitored CA II esterase activity against the substrate 4-nitrophenyl acetate in a format allowing high-throughput screening. The assay proved to be robust and reproducible with a hit rate of approximately 2%. Potential hits were further characterized by determining their IC(50) and K(d) values and tested for nonspecific, promiscuous inhibition. Three known sulfonamide CA inhibitors were identified: acetazolamide, methazolamide, and celecoxib. Other hits were also found, including diuretics and antibiotics not previously identified as CA inhibitors, for example, furosemide and halazone. These results confirm that many sulfonamide drugs have CA inhibitory properties but also that not all sulfonamides are CA inhibitors. Thus many, but not all, sulfonamide drugs appear to interact with CA II and may target other CA isozymes. The screen also yielded several novel classes of nonsulfonamide inhibitors, including merbromin, thioxolone, and tannic acid. Although these compounds may function by some nonspecific mechanism (merbromin and tannic acid), at least 1 (thioxolone) appears to represent a genuine CA inhibitor. Thus, this study yielded a number of potentially new classes of CA inhibitors and preliminary experiments to characterize their mechanism of action.     

Inhibition of malate dehydrogenase by thyroxine and structurally related compounds.

The inhibition of pig heart mitochondrial malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37) by the thyroxine and structurally related compounds was studied to resolve a longstanding question about the exact nature of the inhibition. Thyroxine, in freshly prepared solution, was found to be a "pure" competitive inhibitor relative to the nucleotide cofactor. Upon standing in diffuse daylight, solutions of thyroxine showed increased ability to inhibit the enzyme, presumably as a result of oxidation of enzyme sulfhydryl groups by free iodine that is released photochemically. This behavior probably accounts for earlier reports of irreversible inactivation by thyroxine. Comment is made on the implications of these findings to the mechanism of thyroid hormmone action.     

Deacylation of structurally diverse lipopolysaccharides by human acyloxyacyl hydrolase

Acyloxyacyl hydrolase, a leukocyte enzyme previously has been shown to catalyze the hydrolysis of secondary (acyloxyacyl-linked) fatty acyl chains from the nonreducing glucosamine of the lipid A region of rough Salmonella typhimurium lipopolysaccharide (LPS). We describe here the activity of this enzyme toward smooth S. typhimurium LPS and LPS from Escherichia coli, Pseudomonas aeruginosa, Haemophilus influenzae, Neisseria meningitidis, and Neisseria gonorrhoeae. Acyloxyacyl hydrolase released the secondary acyl chains from all of these lipopolysaccharides, regardless of the location of the acyloxyacyl linkage on the diglucosamine backbone or the structure of the acyl chains. The two acyloxyacyl linkages present in each LPS molecule apparently were hydrolyzed separately, so that free fatty acids released from the different sites accumulated at different rates. The purified enzyme also removed greater than 90% of the secondary acyl chains in each LPS, indicating that the enzyme acts not only on intact LPS but also on LPS molecules that have only one secondary acyl chain. The enzyme did not release the glucosamine-linked 3-hydroxyacyl chains. The specificity and versatility of the enzyme for cleaving acyloxyacyl linkages suggest that it may be a useful reagent for studying the structure and bioactivities of lipopolysaccharides with diverse carbohydrate and lipid A structures.     

Uncoupling and specific inhibition of phosphoryl transfer reactions in mitochondria by antibiotic A20668.

A20668 A, B, and C are polypeptide antibiotics that inhibit phosphorylation of ADP, Mg2t-ATPase, and the ATP-driven transhydrogenase of rat liver submitochondrial particles, but not the purified F1 ATPase. In intact mitochondria, 120668 inhibits uncoupler-induced ATPase, State 3 respiration, and phosphorylation; the A and B forms are approximately equipotent with rutamycin, whereas A20668 C is less effective. Concentrations of A20668 slightly greater than required for complete inhibition of phosphoryl transfer stimulate rapid, uncoupled respiration by mitochondria under State 3 of 4 conditions. A20668 A and B are more effective uncouplers than A20668 C. In the presence of venturicidin or ossamycin, concentrations of A20668, which alone do not uncouple, stimulate oxygen consumption of mitochondria incubated under either State 3 of 4 conditions. A20668 uncoupling is not potentiated by prior inhibition of phosphoryl transfer by venturicidin X, rutamycin, aurovertin, or efrapeptin. A20668 increases mitochondrial permeability to protons in passive swelling experiments where facilitation of proton conductance correlates well with potency to uncouple. A20668 apparently binds initially at a unique locus to inhibit mitochondrial phosphoryl transfer reactions. When this site is saturated, additional antibiotic may uncouple by increasing proton conductance of mitochondria. Binding of venturicidin or ossamycin appears to interfere with the binding of A20668 to its adjacent inhibitory site, thus effectively increasing the concentration of A20668 available to uncouple.     

Cytochrome P-450 cholesterol 7 alpha-hydroxylase: inhibition of enzyme deactivation by structurally diverse calmodulin antagonists and phosphatase inhibitors

Cytochrome P-450 cholesterol 7 alpha-hydroxylase (P-450Ch7 alpha) catalyzes the first and rate-limiting step in the conversion of cholesterol to bile acids. Incubation of rat liver microsomes in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer resulted in a time-dependent deactivation of P-450Ch7 alpha which was markedly accelerated by the nonionic detergent Tween 80. Microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450-dependent 7-ethoxycoumarin O-deethylase activities were unaffected under these conditions, evidencing the selectivity of the deactivation process for P-450Ch7 alpha. The rate (t 1/2 = 15-19 min at 37 degrees C) and maximal extent of P-450Ch7 alpha deactivation (greater than or equal to 90%) were both unaffected by the presence of cytosolic proteins and were also not dependent on the initial enzyme level, as shown using liver microsomes isolated from untreated, cholestyramine-fed, and xenobiotic-induced rats exhibiting an eight-fold range in P-450Ch7 alpha activity. Scavengers for reduced oxygen species were also without effect. P-450Ch7 alpha was stabilized some six- to sevenfold (t 1/2 = 94-143 min) by the phosphatase inhibitor NaF. Of a series of other phosphatase inhibitors examined, including, among others, EDTA, vanadate, and molybdate, only phosphate-containing compounds and the calmodulin antagonist trifluoperazine, and inhibitor of the Ca2+-calmodulin-dependent phosphatase calcineurin, effectively stabilized P-450Ch7 alpha. Modulation of P-450Ch7 alpha deactivation by these inhibitors generally paralleled their effects on isolated calcineurin. A variety of structurally diverse calmodulin antagonists examined were also found to effectively protect P-450Ch7 alpha from deactivation; these include calmidazolium and tamoxifen (IC50 = 25 to 50 microM), chlorpromazine, thioridazine, amitriptyline, imipramine, and the naphthalene sulfonamide compound W-7 (IC50 = 50 to 300 microM). Structure-activity analysis of several phenothiazines and their derivatives indicated that although little activity was exhibited by the sulfoxides, some protection was provided by the corresponding sulfones. On the basis of these observations, various models for the molecular basis of enzyme deactivation are considered, including the hypothesis that a calcineurin-like microsomal phosphatase mediates deactivation of this cytochrome P-450 enzyme.